T cell immune homeostasis is a balance between activation and regulation. In the initial period of funding, we performed detailed studies of T cell activation at a single cell level. In this renewal, we now will focus on the "other half' of the picture, T cell regulation. Our aims will pursue new preliminary data on IL-2R signal transduction in CD4+CD25+ suppressor T cells, and the potential role of the lipid phosphatase PTEN in regulating this pathway. CD4+CD25+ suppressor T cells are essential for homeostasis of the immune system. Strikingly, unlike effector CD4+ T cells, CD4+CD25+ T regs fail to proliferate in response to IL-2 stimulation alone, although they express all 3 chains of the IL-2R. Our data show that CD4+CD25+ T regs are responsive to IL-2 by: (1) undergoing blast transformation without cell division; and (2) acquiring resistance to apoptosis with induction of Bcl-xL protein. Detailed biochemical studies of IL-2R signal transduction in CD4+CD25+ T regs demonstrate intact and normal phosphorylation of STAT-5 but absence of phosphorylation of the PI-3K targets Akt or p70S6 kinase. Surprisingly, we find that PI-3K itself is activated by IL-2, suggesting downstream regulation of PI-3K targets in Tregs. Additional data demonstrate that Tregs constitutively express high levels of the lipid phosphatase PTEN, a regulation of PI-3K signal transduction, and that TCR stimulation of Tregs, which is known to induce IL-2 responsiveness, completely blocks PTEN expression in association and newly enables IL-2 mediated PI-3K downstream signaling. These data show that IL-2R signaling in T regs is distinctly different from that of =normal" T effector cells, and strongly implicates PTEN as an integral part of that process. Based on these data we have three aims: In aim #1, we will determine the kinetics of PTEN downregulation following TCR activation. Pharmacologic inhibitors as well as mice and cell lines with defects in TCR signaling will next be used to determine the signaling mechanisms which regulate this process. In aim #2 we will use mice with partial and complete deficiencies of PTEN in T cells, as well as with inducible deficiencies, to determine the role of PTEN in regulatory cell development and in vitro function, in particular on IL-2 requirements and responsiveness. Finally, in aim #3, we will employ an autoimmune and a transplant model to study the in vivo role of PTEN in Treg biology.